Highly efficient editing of the ß-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease

So Hyun Park , Ciaran M Lee , Daniel P Dever , Timothy H Davis , Joab Camarena , Waracharee Srifa , Yankai Zhang , Alireza Paikari , Alicia K Chang , Matthew H Porteus , Vivien A Sheehan , Gang Bao 
Source: Nucleic Acids Res
Publication Date: (2019)
Issue: 47(15): 7955-7972
Research Area:
Immunotherapy / Hematology
Regenerative medicine
Cells used in publication:
Species: human
Tissue Origin: blood
CD34+ cell, human
Species: human
Tissue Origin: blood
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector™ X-Unit

A total of 2 × 105 K562 cells (program FF-120, solution SF), U2OS cells (program CM-137, solution SE) and CD34+ cells (program CA-137, solution P3) were electroporated on a Lonza Nucleofector 4-D according to manufacturer’s instructions. In K562 cells, 1µg of pX330 plasmid and 0–5 µM (0–100 pmol) ssODN template (Ultramer ®DNA Oligonucleotides from Integrated DNA Technologies) were transfected. InU2OS, 1 µg of pX330 plasmid and 100 pmol of dsODN were transfected for GUIDE-seq analysis. In CD34+ cells, 5 µg (30.5 pmol) of Cas9 protein (Feldan Therapeutics or Integrated DNA Technologies), 2.5 µg (73 pmol) of chemically synthesized gRNAs (TriLink BioTechnologies) and 0–5 µM of ssODN were transfected. For mock-treated CD34+ cells, electroporation of the same amount of cellswas performed using the same P3 buffer and CA-137 program as the treated cells, but without RNP or ssODN.


Sickle cell disease (SCD) is a monogenic disorder that affects millions worldwide. Allogeneic hematopoietic stem cell transplantation is the only available cure. Here, we demonstrate the use of CRISPR/Cas9 and a short single-stranded oligonucleotide template to correct the sickle mutation in the ß-globin gene in hematopoietic stem and progenitor cells (HSPCs) from peripheral blood or bone marrow of patients with SCD, with 24.5 ± 7.6% efficiency without selection. Erythrocytes derived from gene-edited cells showed a marked reduction of sickle cells, with the level of normal hemoglobin (HbA) increased to 25.3 ± 13.9%. Gene-corrected SCD HSPCs retained the ability to engraft when transplanted into non-obese diabetic (NOD)-SCID-gamma (NSG) mice with detectable levels of gene correction 16-19 weeks post-transplantation. We show that, by using a high-fidelity SpyCas9 that maintained the same level of on-target gene modification, the off-target effects including chromosomal rearrangements were significantly reduced. Taken together, our results demonstrate efficient gene correction of the sickle mutation in both peripheral blood and bone marrow-derived SCD HSPCs, a significant reduction in sickling of red blood cells, engraftment of gene-edited SCD HSPCs in vivo and the importance of reducing off-target effects; all are essential for moving genome editing based SCD treatment into clinical practice.